The present invention relates to nucleic acid polymorphisms and, in particular, relates to detecting a single nucleotide polymorphism using nucleic acid amplification technology.
Studies designed to determine the sequence of the human genome, as well as studies designed to compare human genomic sequences, have elicited information regarding polymorphisms in the human genome. A wide variety of polymorphisms in the human genome have previously been described. The various types of human genetic polymorphisms include single base substitutions; insertions or deletions; variable numbers of tandem repeats; deletions of all or a large part of a gene; gene amplifications; and chromosomal rearrangements. Generally, polymorphisms that involve a single nucleotide are called single nucleotide polymorphisms (xe2x80x9cSNPsxe2x80x9d).
Recently, a SNP in codon 16 of the xcex22 adrenergic receptor gene has been reported and associated with a variation in response to xcex2 agonist therapy (Drazen, J M et al, Thorzx, 1996, 51:1168; Liggett, S. B., Am. J. Respir. Crit. Care Med., 1997, 156:S156-62; Martinez, F. D. et al, J. Clin. Invest., 1997, 100:3184-8). Adrenergic receptors are hormone receptors on the surfaces of various cells. When bound to an adrenergic receptor site, a hormone can trigger a cascade of cellular events. Hence, adrenergic receptors and the hormones that bind to them, in large part form the mechanism that controls cellular events at a molecular level. Many pharmacological compounds mimic molecules that bind to adrenergic receptor sites and, in this manner, clinically regulate cellular function. For example, a class of drugs known as beta-agonists bind to xcex22 adrenergic receptor sites and are widely used as a medication for asthma. Individuals with a SNP in codon 16 of the xcex22 adrenergic gene, however, may not respond to such therapies due to a conformational, or other, change in the receptor that causes a decrease in the affinity between the receptor and the medication or hormone.
It would be advantageous, therefore, to provide a means for detecting the polymorphism in codon 16 of the xcex22 adrenergic receptor gene prior to prescribing medications that would not be efficacious as a result of the polymorphism.
Provided herein are methods capable of analyzing polymorphic nucleic acid sequences in a manner suitable for automation. The present invention provides reagents, methods, and kits for amplifying and detecting a target sequence having a polymorphism at codon 16 of the xcex22 adrenergic receptor gene in a test sample. In particular, SEQ ID NO: 2 and SEQ ID NO: 3 can be employed as amplification primers to amplify the target sequence designated herein as SEQ ID NO: 1. It was discovered that these primers specifically and sensitively produce an amplification product that is amenable to detection with SEQ ID NOs: 4, 5, and with molecular beacon probes comprising SEQ ID NOs: 6, 7, 8, and 9. SEQ ID NO: 4 is an internal hybridization probe specific for the wild-type sequence and SEQ ID NO: 5 is an internal hybridization probe specific for the variant (polymorphic) sequence. Similarly, SEQ ID NOs: 6 and 7 are the nucleotide sequence incorporable into a molecular beacon probe for the wild-type sequence, while SEQ ID NOs: 8 and 9 are nucleotide sequences incorporable into molecular beacon probes selective for the variant (polymorphic) sequences.
The target sequence, designated herein as SEQ ID NO: 1, can be amplified by forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing a target sequence, and primers designated SEQ ID NOs. 2 and 3. Following amplification, the amplified target sequence can be detected. For example, the probes designated SEQ ID NOs: 4 and 5, or the molecular beacon probes incorporating the sequences designated SEQ ID NOs: 6, 7, 8 and 9, can be employed to hybridize to the amplified target sequence to form a probe/amplification product hybrid, which can be detected using any suitable technique selected from a variety of well known techniques. Hence, detecting a probe/amplification product hybrid wherein the probe is SEQ ID NO: 4 indicates the presence of the wild-type sequence. On the other hand, detecting of a probe/amplification product hybrid wherein the probe is SEQ ID NO: 5 would indicate the presence of the polymorphic sequence. Similarly, detecting a signal indicative of the target-bound state from the molecular beacon probe comprising the nucleotide sequence designated SEQ ID NO: 6 or SEQ ID NO: 7 in the presence of the amplification product indicates the presence of the wild-type sequence, whereas detecting a signal indicative of the target-bound state from one of the molecular beacon probes comprising the nucleotide sequence designated SEQ ID NO: 8 or SEQ ID NO: 9 in the presence of the amplification product indicates the presence of the variant (polymorphic) sequence.
The present invention provides reagents, methods, and kits for amplifying and detecting a target sequence in a test sample. In particular, SEQ ID NO: 2 and SEQ ID NO: 3 can be employed as amplification primers to amplify a nucleic acid sequence potentially comprising the polymorphism in codon 16 of the xcex22 adrenergic receptor gene. Hence, both the wild-type and polymorphic version of the target sequence can be amplified using SEQ ID NOs:2 and 3. The sequence AACGGCAGCG CCTTCTTGCT GGCACCCAAT AGAAGCCATG CGCCGGACCA CGACGTCACG CAGCAAAGGG ACGAGGTGTG GGTGGTGGGC ATGGGCATCG TCATGT (SEQ ID NO: 1) is presented as a representative target sequence. Probe sequences, having SEQ ID NO: 4 or SEQ ID NO: 5 can be employed to detect or distinguish the amplification product produced by primers designated SEQ ID NO: 2 and SEQ ID NO: 3 (e.g., indicate the presence of the wild-type or polymorphic sequence in the test sample). Similarly, molecular beacon probes incorporating the nucleotide sequences designated SEQ ID NOs: 6, 7, 8, and 9 can be employed to detect and/or distinguish the amplification product produced by primers designated SEQ ID NOs: 2 and 3 (e.g., indicate the presence of the wild-type or polymorphic sequence in the test sample).
Nucleotide sequences useful in the context of the present invention include:
as well as artificial analog sequences wherein one or more of the naturally-occurring nucleotides identified above is replaced with a synthetic analog of the nucleotide. In three preferred embodiments, the molecular beacons comprising the nucleotide sequences designated SEQ ID NOs: 6, 7, 8, and 9 consist entirely or essentially of these nucleotide sequences, linked to fluorescein and dabcyl. Molecular beacon probes comprising the nucleotides sequences designated SEQ ID NO: 6 and SEQ ID NO: 9 are preferred to molecular beacon probes comprising SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
The primer, probe, and nucleotide sequences of the molecular beacons disclosed herein, can comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs, such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference, as well as other nucleic acid analogs known in the art. For example, the skilled artisan will recognize that where the oligonucleotide designated as T (i.e., thymidine) is indicated, this oligonucleotide also designates U in embodiments where RNA is employed rather than DNA, and designates a nucleic acid analog where non-naturally occurring nucleotide residues are incorporated into the sequences of the present invention. (Such sequences routinely can be synthesized using a variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, Calif.); DuPont, (Wilmington, Del.); or Milligen, (Bedford, Mass.). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882, each of which are herein incorporated by reference in their entirety. In certain embodiments, the probes and molecular beacon probes of the present invention preferably contain only the naturally-occurring RNA nucleotides (i.e., A, G, C, and U), and more preferably, contain only the naturally-occurring DNA nucleotides (i.e., A, G, C, and T). Sequences employed as primers preferably have DNA at the 3xe2x80x2 end of the sequence, and more preferably, are completely comprised of DNA.
A xe2x80x9ctarget sequencexe2x80x9d is a nucleic acid sequence that is both amplified and detected, or comprises a nucleotide sequence complementary to SEQ ID NO: 1. While the term target sequence is sometimes referred to as single stranded, the skilled artisan will recognize that the target sequence as used herein can be double stranded.
The term xe2x80x9ctest samplexe2x80x9d as used herein, means anything suspected of containing the target sequence. The test sample can be derived from any biological source, such as for example, blood, bronchial alveolar lavage, saliva, throat swabs, ocular lens fluid, cerebral spinal fluid, sweat, sputa, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissues such as heart tissue and the like, or fermentation broths, cell cultures, chemical reaction mixtures and the like. The test sample can be used (i) directly as obtained from the source or (ii) following a pre-treatment to modify the character of the sample. Thus, the test sample can be pre-treated prior to use by, for example, preparing plasma from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, purifying nucleic acids, and the like. Most typically, the test sample will be whole blood.
SEQ ID NOs: 2 and 3 can be used as amplification primers according to amplification procedures well known in the art to amplify the target sequence. Preferably, the sequences provided herein are employed according to the principles of the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202 which are herein incorporated by reference. It will be understood by those skilled in the art that in the event that the target sequence is RNA, a reverse transcription step can be included in the amplification of the target sequence. Enzymes having reverse transcriptase activity are well known for their ability to produce a DNA sequence from an RNA template. Reverse transcription PCR (RT PCR) is well known in the art and described in U.S. Pat. Nos. 5,310,652 and 5,322,770, which are herein incorporated by reference.
Thus, amplification methods of the present invention generally comprise the steps of forming a reaction mixture comprising nucleic acid amplification reagents, amplification primers (i.e., SEQ ID NO: 2 and SEQ ID NO: 3), and a test sample suspected of containing a target sequence. Upon formation of the reaction mixture, the so-formed reaction mixture is subjected to amplification conditions to generate at least one copy of the target sequence. It will be understood that subjecting the reaction mixture may be repeated several times such as by thermal cycling the reaction mixture as is well known in the art.
As stated above, the reaction mixture comprises xe2x80x9cnucleic acid amplification reagentsxe2x80x9d that include reagents which are well known and may include, but are not limited to, an enzyme having polymerase activity (and, as necessary, reverse transcriptase activity), enzyme cofactors such as magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as for example deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
xe2x80x9cAmplification conditionsxe2x80x9d are defined generally as conditions which promote hybridizing or annealing of primer sequences to a target sequence and subsequent extension of the primer sequences. It is well known in the art that such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, sequence length, complementarity, and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. For any given set of sequences, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures which are close to (i.e., within 10xc2x0 C.) the melt temperature. Ionic strength or xe2x80x9csaltxe2x80x9d concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by interacting with the phosphodiester backbone. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased sequence length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer sequences have more hydrogen bonds holding the sequences together. Thus, a high G:C content and longer sequence lengths impact the hybridization conditions by elevating the melt temperature.
Once sequences are selected for a given diagnostic application, the G:C content and length will be known and can be accounted for in determining precisely what hybridization conditions will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature. Generally, the hybridization temperature is selected close to or at the Tm of the primers or probe. Thus, obtaining suitable hybridization conditions for a particular primer, probe, or primer and probe set is well within ordinary skill of one practicing this art.
Amplification products produced as above can be detected during or subsequently to the amplification of the target sequence. Detection platforms that can be employed to detect the amplification products produced with SEQ ID NOs: 2 and 3 using probe sequences having SEQ ID NOs: 4 and 5 or molecular beacon probes comprising SEQ ID NOs: 6, 7, 8, and/or 9, include any of the well known homogeneous or heterogeneous techniques well known in the art. Examples of homogeneous detection platforms can include the use of Fluorescence Resonance Energy Transfer (FRET) labels attached to probes that emit a signal in the presence of the target sequence, such as, without limitation, fluorescein and dabcyl. So-called TaqMan assays described in U.S. Pat. No. 5,210,015 (herein incorporated by reference) and molecular beacon probes and assays described in U.S. Pat. No. 5,925,517 (herein incorporated by reference) are examples of techniques that can be employed to homogeneously detect nucleic acid sequences. Additionally, such platforms can be employed to detect the production of amplification product in a real-time manner. It will be understood that the probes can be modified to such that they are suitable for use according to the particular detection platform employed.
Gel electrophoresis, for example, can be employed to detect the products of an amplification reaction after its completion using molecular weight markers. However, amplification products can be detected heterogeneously using labeled probes and solid supports. Hence, methods for detecting the amplified target sequence can include the steps of (a) hybridizing at least one hybridization probe (e.g., SEQ ID NOs: 4 and 5) to the nucleic acid sequence complementary to the target sequence, so as to form a hybrid comprising the probe and the nucleic acid sequence complementary to the probe; and (b) detecting the hybrid as an indication of the presence of the target sequence in the test sample.
Hybrids formed as above can be detected using microparticles and labels that can be used to separate and detect such hybrids. Preferably, detection is performed according to the protocols used by the commercially available Abbott LCx(copyright) instrumentation (Abbott Laboratories; Abbott Park, Ill.).
Similarly, methods for detecting the amplified target sequence include the steps of (a) contacting a molecular beacon probe comprising a nucleic acid having the sequence designated SEQ ID NO: 6, 7, 8, or 9 with a target sequence or amplification product under suitable conditions, and (b) observing the emission spectrum or spectra of the molecular beacon to determine whether or how much of the target or amplification product is present.
Molecular beacon probes comprising SEQ ID NOs: 6, 7, 8, and/or 9 also can be used to detect the presence or quantity of the amplification product in other embodiments. For example, molecular beacon probes can be added to the amplification reaction, thereby allowing essentially simultaneous amplification and detection of the amplification products in a single reaction. As another alternative, the amplification products can be isolated and then contacted with molecular beacon probes comprising SEQ ID NOs: 6, 7, 8, and/or 9.
The term xe2x80x9clabelxe2x80x9d as used herein means a molecule or moiety having a property or characteristic which can be detected. A label can be directly detectable, as with, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly detectable, as with, for example, specific binding members. Directly detectable labels may require additional components such as, for example, substrates, triggering reagents, light, and the like to enable detection of the label. When indirectly detectable labels are used, they are typically used in combination with a xe2x80x9cconjugatexe2x80x9d. A conjugate is typically a specific binding member that has been attached or coupled to a directly detectable label. Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of the specific binding member or the detectable property of the label. As used herein, xe2x80x9cspecific binding memberxe2x80x9d means a member of a binding pair, i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule. In addition to antigen and antibody specific binding pairs, other specific binding pairs include, but are not intended to be limited to, avidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors or substrates and enzymes; and the like.
A molecular beacon probes as used herein comprises at least two moieties to form at least one label. A molecular beacon is a moiety comprising a first chemical moiety conjugated to a nucleic acid or nucleic acid analog that is conjugated to a second chemical moiety. (See, e.g., Tyagi et al., Nat. Biotechnol., 14, 303-308 (1996), and U.S. Pat. Nos. 5,119,801 and 5,312,728, each of which is incorporated by reference). The nucleic acid, or nucleic acid analog, is capable of binding to itself to form a stem-and-loop structure, which brings the first chemical moiety into close proximity to the second chemical moiety. When in close proximity, the first chemical moiety, or the second chemical moiety, or the combination of the chemical moieties either produce no detectable signal, or in the alternative produce an xe2x80x9cunbound signalxe2x80x9d that is characteristic of the molecular beacon when it is not bound to the target or amplification product. The first chemical moiety and second chemical moiety are each preferably located at opposite termini of the nucleic acid sequence, but can also be incorporated into internal positions within the nucleic acid.
Conversely, when the molecular beacon is contacted with the target or amplification product, the molecular beacon probe binds with the target and/or amplification product. Binding of the molecular beacon to the target or amplification product forces the molecular beacon into a conformation that separates the first chemical moiety and the second chemical moiety of the molecular beacon. In this separated state, the molecular beacon generates a xe2x80x9cbound signalxe2x80x9d that can be distinguished from the xe2x80x9cunbound signal.xe2x80x9d The bound signal can be generated by the first chemical moiety, the second chemical moiety, by both moieties, or can be the absence of the unbound signal.
The first chemical moiety and the second chemical moiety can be any suitable atom or molecular moiety capable of detection. Preferred embodiments of the first chemical moiety and second chemical moiety of the molecular beacon probe include any suitable fluorophore. The first chemical moiety and second chemical moiety preferably are capable of interacting via Fluorescence Resonance Energy Transfer (FRET) or through collisional quenching.
FRET is a form of molecular energy transfer by which energy is passed between a donor molecule and an acceptor molecule. FRET arises from the properties of certain chemical compounds; when excited by exposure to particular wavelengths of light, they emit light (i.e., they fluoresce) at a different wavelength. Such compounds are termed fluorophores. In FRET, energy is passed non-radiatively over a long distance between a donor molecule, which is a fluorophore, and an acceptor molecule. The donor absorbs a photon and transfers energy nonradiatively to the acceptor (reviewed in Clegg, Methods Enzymology, 211, 353-388 (1992)).
Depending on what kind of fluorophore molecule is being monitored (i.e. a donor or an acceptor), the fluorescence is either quenched or enhanced by a transfer of energy. Resonant overlap of the excitation and emission spectra of two fluorophores enables an energy transfer. Energy transfer also depends on physical factors, such as the orientation and distance between the two fluorophores. Resonance energy transfer is well known in the art, and skilled workers are able to choose compatible pairs for fluorescence quenching or enhancement, along with useful excitation and fluorescence detection wavelengths. The disclosures of U.S. Pat. Nos. 5,691,146; 5,876,930; 5,723,591; 5,348,853; 5,119,801; 5,312,728; 5,962,233; 5,942,283; 5,866,336, discussing such fluorophores, are incorporated herein by reference.
Whether a fluorophore is a donor or an acceptor is defined by its excitation and emission spectra, and that of the fluorophore with which it is paired. For example, FAM is most efficiently excited by light with a wavelength of 488 nm, and emits light with a spectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM is a suitable donor fluorophore for use with JOE, TAMRA, and ROX (all of which have excitation maximum at 514 nm).
Molecules that are commonly used in FRET include Fluorescein, 5-carboxyfluorescein (FAM), 2xe2x80x27xe2x80x2 dimethoxy-4xe2x80x25xe2x80x2-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4xe2x80x2-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2xe2x80x2-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).
Non-FRET based molecular beacon probes, such as but not limited to collisional quenching pairs, are also preferred in the context of the present invention. Collisional quenching pairs are described in U.S. Pat. No. 6,150,097, which is hereby incorporated.
Other suitable labels are known in the art, and can readily be selected by the skilled artisan, irrespective of whether the label is to be used in FRET.
A xe2x80x9csolid supportxe2x80x9d, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. Thus, a solid support can be can be latex, plastic, derivatized plastic, magnetic or non-magnetic metal, glass, silicon or the like. A vast array of solid support configurations are also well known and include, but are not intended to be limited to, beads, shavings, grains, particles, plates, or tubes.
According to one embodiment, hybrids can be detected by incorporating labels in the primer and/or probe sequences to facilitate detection. Hence, first and second specific binding members attached to the primers and probes can be employed to immobilize the hybrids to, for example, microparticles and detect the presence of the hybrids on the microparticles with the assistance of a conjugate.
According to another embodiment, a combination of specific binding members and directly detectable labels can be employed to detect hybrids. For example, specific binding members can be introduced in the hybrids using primers labeled with specific binding members. A directly detectable label can be incorporated into the hybrids using a probe that has been labeled with a directly detectable label. Hence, hybrids can be immobilized to a microparticle using the specific binding member and directly detected by virtue of the label on the probe. It will be understood that other detection configurations are a matter of choice for those skilled in the art.
According to a preferred embodiment, xe2x80x9coligonucleotide hybridization PCRxe2x80x9d (variably referred to herein as xe2x80x9cOH PCRxe2x80x9d) amplification reaction as described in U.S. patent application Ser. No 08/514,704, filed Aug. 14, 1995, now abandoned, that is herein incorporated by reference, is employed to detect the target sequence. Briefly, the reagents employed in the preferred method comprise at least one amplification primer and at least one internal hybridization probe, as well amplification reagents for performing an amplification reaction. The primer sequence is employed to prime extension of a copy of a target sequence (or its complement) and is labeled with either a capture label or a detection label. The probe sequence is used to hybridize with the sequence generated by the primer sequence, and typically hybridizes with a sequence that does not include the primer sequence. Similarly to the primer sequence, the probe sequence is also labeled with either a capture label or a detection label with the caveat that when the primer is labeled with a capture label the probe is labeled with a detection label and vice versa. Detection labels have the same definition as xe2x80x9clabelsxe2x80x9d previously defined and xe2x80x9ccapture labelsxe2x80x9d are typically used to separate extension products, and probes associated with any such products, from other amplification reactants. Specific binding members (as previously defined) are well suited for this purpose. Also, probes used according to this method are preferably blocked at their 3xe2x80x2 ends so that they are not extended under hybridization conditions. Methods for preventing extension of a probe are well known and are a matter of choice for one skilled in the art. Typically, adding a phosphate group to the 3xe2x80x2 end of the probe will suffice for purposes of blocking extension of the probe.
According to the above preferred embodiment the probe initially is part of the reaction mixture, it is preferable to select primers, probes and amplification conditions such that the probe sequence has a lower melt temperature than the primer sequences so that upon placing the reaction mixture under amplification conditions copies of the target sequence or its complement are produced at temperature above the Tm of the probe. After such copies are synthesized, they are denatured and the mixture is cooled to enable the formation of hybrids between the probes and any copies of the target or its complement. The rate of temperature reduction from the denaturation temperature down to a temperature at which the probes will bind to single stranded copies is preferably quite rapid (for example 8 to 15 minutes) and particularly through the temperature range in which an enzyme having polymerase activity is active for primer extension. Such a rapid cooling favors copy sequence/probe hybridization rather that primer/copy sequence hybridization and extension.
The following examples further illustrate the present invention and are not intended to limit the invention in any way.